Method of preventively treating a subject at the risk of developing infections of a respiratory virus

ABSTRACT

A method of preventively treating a subject at the risk of developing infections of a respiratory virus is disclosed. The method includes a step of administering to the subject an effective amount of a peptide synthesized through a chemical route or by a genetic engineering process, characterized in that the peptide has a functional domain capable of binding to a surface glycoprotein of a respiratory virus and has an activity of inhibiting infection of the respiratory virus, wherein the peptide has 5 or more basic amino acids, among which 2 or more basic amino acids are in N-terminal region or C-terminal region of the peptide; and wherein the peptide consists of an amino acid sequence that is at least 90% identical to SEQ ID NO: 10. The invention also discloses the mechanism of the peptides in inhibition of said infections, which provides theory support for developing new prophylactic/therapeutic agents with broad-spectrum antiviral activities.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 13/999,393 filed on Feb. 21, 2014 which claims the benefit offiling of U.S. provisional application No. 61/821,292 filed on May 9,2013 and claims priority to Chinese Patent Application No.201310451941.2, filed on Sep. 27, 2013, the disclosure of all of theseapplications is incorporated herein, in its entirety, by reference.

FIELD OF THE INVENTION

This invention relates to the field of anti-viral prophylaxis andtherapy. In particular, the invention relates to a peptide havingactivity of inhibiting infections of respiratory viruses, a method ofblocking infections of respiratory viruses in target cells using thepeptide, a method of therapeutically or preventively treating a subjectinfected or at the risk of developing infections of respiratory virusesusing the peptide, a composition comprising the peptide, as well as akit for screening a peptide capable of inhibiting infections ofrespiratory viruses and the screening method.

BACKGROUND OF THE INVENTION

Antimicrobial peptides (AMPs) are intrinsic host defence molecules whichare probably produced by all multicellular plants and animals (see“Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature415, 389-395 (2002)”). They comprise the first line of innate immunesystem to rapidly eliminate invading pathogens in the early stage ofinfection and can also promote systemic adaptive immune response (see“Rohrl, J., Huber, B., Koehl, G. E., Geissler, E. K. & Hehlgans, T.Mouse beta-defensin 14 (Defb14) promotes tumor growth by inducingangiogenesis in a CCR6-dependent manner. J Immunol 188, 4931-4939(2012)” and “Yang, D., et al. Beta-defensins: linking innate andadaptive immunity through dendritic and T cell CCR6. Science 286,525-528 (1999)”). Most AMPs are amphipathic and cationic molecules,which confer the binding ability to the microbe membranes that aregenerally negatively charged. Many hundreds of AMPs have been identifiedand classified according to their structural features and/or amino acidcompositions. Two families of AMPs in vertebrates, cathelicidins anddefensins, are small molecules mainly produced by leucocytes andepithelia cells (see “Lehrer, R. I. Primate defensins. Nat Rev Microbiol2, 727-738 (2004)” and “Selsted, M. E. & Ouellette, A. J. Mammaliandefensins in the antimicrobial immune response. Nat Immunol 6, 551-557(2005)”). The precursors of cathelicidins contain a conservedamino-terminal “cathelin” domain (about 100-residue-long). Processedcathelicidin peptides range in length from 12 to 80 amino acid residues,with or without α-helical, β-sheet or other types of tertiary structures(see “Lehrer, R. I. & Ganz, T. Cathelicidins: a family of endogenousantimicrobial peptides. Curr Opin Hematol 9, 18-22 (2002)” and “Zanetti,M. Cathelicidins, multifunctional peptides of the innate immunity. JLeukoc Biol 75, 39-48 (2004)”). Defensins are small (2-6 kD)cysteine-rich AMPs which mainly form β-sheet structures stabilized bythree (rarely four) conserved intramolecular disulphide bridges. Threesubfamilies of defensins are further classified as α-, β- andθ-defensins in vertebrates according to their disulfide patterns (see“Lehrer, R. I. Primate defensins. Nat Rev Microbiol 2, 727-738 (2004)”).These peptides generally have a broader range of non-specific activityagainst infections of microorganisms, including gram-positive andgram-negative bacteria, fungi and viruses. The diverse action modes ofAMPs against bacteria include disrupting membrane integrity (see “Shai,Y. Mechanism of the binding, insertion and destabilization ofphospholipid bilayer membranes by alpha-helical antimicrobial and cellnon-selective membrane-lytic peptides. Biochim Biophys Acta 1462, 55-70(1999)” and “Yang, L., Weiss, T. M., Lehrer, R. I. & Huang, H. W.Crystallization of antimicrobial pores in membranes: magainin andprotegrin. Biophys J 79, 2002-2009 (2000)”), impairing nucleus andprotein synthesis, inhibiting chaperone-assisted protein fold,interrupting cell-wall biosynthesis pathway and targeting membranebiogenesis (see “Brogden, K. A. Antimicrobial peptides: pore formers ormetabolic inhibitors in bacteria? Nat Rev Microbiol 3, 238-250 (2005)”,“Hale, J. D. & Hancock, R. E. Alternative mechanisms of action ofcationic antimicrobial peptides on bacteria. Expert Rev Anti Infect Ther5, 951-959 (2007)” and “Srinivas, N., et al. Peptidomimetic antibioticstarget outer-membrane biogenesis in Pseudomonas aeruginosa. Science 327,1010-1013 (2010)”). Thus far, however, the antiviral mechanism of AMPsis still largely unknown.

Defensins have been shown to possess many properties, includingantibacterial (see “Nizet, V., et al. Innate antimicrobial peptideprotects the skin from invasive bacterial infection. Nature 414, 454-457(2001)” and “Mygind, P. H., et al. Plectasin is a peptide antibioticwith therapeutic potential from a saprophytic fungus. Nature 437,975-980 (2005)”), antiviruses (see “Gong, T., et al. Recombinant mousebeta-defensin 2 inhibits infection by influenza A virus by blocking itsentry. Arch Virol 155, 491-498 (2010)” and “Leikina, E., et al.Carbohydrate-binding molecules inhibit viral fusion and entry bycrosslinking membrane glycoproteins. Nat Immunol 6, 995-1001 (2005)”)and antifugi (see “Krishnakumari, V., Rangaraj, N. & Nagaraj, R.Antifungal activities of human beta-defensins HBD-1 to HBD-3 and theirC-terminal analogs Phd1 to Phd3. Antimicrob Agents Chemother 53, 256-260(2009)” and “Jiang, Y., et al. Antifungal activity of recombinant mousebeta-defensin 3. Lett Appl Microbiol 50, 468-473 (2010)”).

Native defensins are produced by innate and adaptive immune systems inresponse to infections, such as viral infection (see “Zasloff, M.Antimicrobial peptides of multicellular organisms. Nature 415, 389-395(2002)”). In mice, gene expression of murine β-defensin-3 andβ-defensin-4 is induced in upper respiratory tract by influenza virusinfection (see “Chong, K. T., Thangavel, R. R. & Tang, X. Enhancedexpression of murine beta-defensins (MBD-1, -2, -3, and -4) in upper andlower airway mucosa of influenza virus infected mice. Virology 380,136-143 (2008)”). HIV-1 Tat can induce human β-defensin-2 expression inhuman B cells (see “Ju, S. M., et al. Extracellular HIV-1 Tat induceshuman beta-defensin-2 production via NF-kappaB/AP-1 dependent pathwaysin human B cells. Mol Cells 33, 335-341 (2012)”).

The induced defensins play important roles in the protection againstinvading microbes in the early stage of infection (see “Zasloff, M.Antimicrobial peptides of multicellular organisms. Nature 415, 389-395(2002)”). Human α-defensin-1 has been demonstrated to inhibit influenzavirus replication, which may be due to attenuation of protein kinase Cactivation (see “Salvatore, M., et al. alpha-Defensin inhibits influenzavirus replication by cell-mediated mechanism(s). J Infect Dis 196,835-843 (2007)”). Mouse β-denfensin-2 has been demonstrated to inhibitviral infection by blocking viral entry into target cells (see “Gong,T., et al. Recombinant mouse beta-defensin 2 inhibits infection byinfluenza A virus by blocking its entry. Arch Virol 155, 491-498(2010)”). A θ-defensin, retrocylin 2 (RC2), was illustrated to inhibitvirus-cell membrane fusion by crosslinking membrane glycoproteins (see“Leikina, E., et al. Carbohydrate-binding molecules inhibit viral fusionand entry by crosslinking membrane glycoproteins. Nat Immunol 6,995-1001 (2005)”). Effectors of adaptive immune system, such asantibodies and T lymphocytes, are highly pathogen specific. In contrast,effectors of innate immune system, such as defensins, generally havebroader spectrum activity against microorganisms. The unique propertiesof defensins make them attractive candidates for development of broaderspectrum antiviral drugs with reduced opportunity of drug-resistance.For antiviral strategies, inhibition of viral entry, viral RNA release,virus replications and release may be selected as the targets fordevelopment of antiviral drugs.

Specific antivirals against common respiratory virus families, such asorthomyxoviridae, paramyxoviridae and coronaviridae causing emerginginfections, are either not available or prone to develop drug-resistancedue to the rapid mutation of these viral genes (see “Cheng, V. C., S. K.Lau, P. C. Woo, and K. Y. Yuen. 2007. Severe acute respiratory syndromecoronavirus as an agent of emerging and reemerging infection. ClinMicrobiol Rev 20:660-694”, “Cheng, V. C., K. K. To, H. Tse, I. F. Hung,and K. Y. Yuen. 2012. Two years after pandemic influenza A/2009/H1N1:what have we learned? Clin Microbiol Rev 25:223-263”, and “Wong, S. S.,and K. Y. Yuen. 2008. Antiviral therapy for respiratory tractinfections. Respirology 13:950-971”). Although many defensins from miceor humans have been found to have antiviral activity in vitro and invivo (see “Jiang, Y., Y. Wang, Y. Kuang, B. Wang, W. Li, T. Gong, Z.Jiang, D. Yang, and M. Li. 2009. Expression of mouse beta-defensin-3 inMDCK cells and its anti-influenza-virus activity. Arch Virol154:639-647”, “Quinones-Mateu, M. E., M. M. Lederman, Z. Feng, B.Chakraborty, J. Weber, H. R. Rangel, M. L. Marotta, M. Mirza, B. Jiang,P. Kiser, K. Medvik, S. F. Sieg, and A. Weinberg. 2003. Human epithelialbeta-defensins 2 and 3 inhibit HIV-1 replication. Aids 17:F39-48”, and“Sun, L., C. M. Finnegan, T. Kish-Catalone, R. Blumenthal, P.Garzino-Demo, G. M. La Terra Maggiore, S. Berrone, C. Kleinman, Z. Wu,S. Abdelwahab, W. Lu, and A. Garzino-Demo. 2005. Human beta-defensinssuppress human immunodeficiency virus infection: potential role inmucosal protection. J Virol 79:14318-14329”), the development ofdefensins as therapeutics has been hindered by several factors, such assuboptimal efficacy, side effects and the lack of cost-effective meansof commercial-scale production.

Thus, a safe, potent and broad-spectrum antiviral is urgently needed tocombat emerging viral respiratory diseases.

SUMMARY OF THE INVENTION Technical Problems to be Solved

One object of the present invention is to provide a peptide havingactivity of inhibiting infections of respiratory viruses. Another objectof the present invention is to provide a composition comprising thepeptide. Still another object of the present invention is to provide amethod of blocking infections of respiratory viruses in target cells anda method of therapeutically or preventively treating a subject infectedor at the risk of developing infections of respiratory viruses. Stillanother object of the present invention is to provide a kit forscreening a peptide capable of inhibiting infections of respiratoryviruses as well as the screening method.

Technical Solutions

Accordingly, the present invention provides a peptide synthesizedthrough a chemical route or by a genetic engineering process, whereinthe peptide has a functional domain capable of binding to a surfaceglycoprotein of a respiratory virus and has an activity of inhibitinginfection of the respiratory virus, wherein the peptide has 5 or morebasic amino acids, among which 2 or more basic amino acids are inN-terminal region or C-terminal region of the peptide; and wherein theN-terminal region comprises a sequence of no more than 10 amino acidscounting from the N-terminal amino acid of the peptide, and theC-terminal region comprises a sequence of no more than 10 amino acidscounting from the C-terminal amino acid of the peptide.

Preferably, the peptide further has a function of preventingacidification in a late endosome of a cell.

Preferably, the peptide has 3 or more basic amino acids in N-terminalregion or C-terminal region thereof.

Preferably, the peptide has 4 or more cysteines.

Preferably, the peptide has an amino acid sequence (a) or (b) asdescribed below:

-   (a) an amino acid sequence as set forth in SEQ ID NO.: 10; or-   (b) an amino acid sequence obtained by substitution, deletion and/or    addition of one or several amino acids in the amino acid sequence    (a).

Preferably, the amino acid sequence (b) is at least 70%, more preferablyat least 80%, further preferably at least 90%, identical to the aminoacid sequence (a).

Preferably, the amino acid sequence of the peptide is as set forth inSEQ ID NO.: 10, SEQ ID NO.: 13, or SEQ ID NO.: 17.

Preferably, the C-terminal region has two cysteines and the basic aminoacids.

Preferably, the C-terminal region has 10 amino acids with the followingamino acid composition:

-   basic amino acid-neutral amino acid-basic amino acid-neutral amino    acid-basic amino acid-cysteine-cysteine-basic amino acid-neutral    amino acid-basic amino acid-free carboxyl.

Preferably, the peptide is originated from human or mouse; and morepreferably, the peptide is originated from mouse β-defensin-4.

Preferably, the respiratory virus is selected from influenza viruses andcoronaviruses; wherein the influenza viruses include influenza virussubtypes H1, H3, H5, and H7, and the coronaviruses include SARS-CoV andMERS-CoV.

It is another aspect of this invention to provide a compositioncomprising: any one of the peptides of this invention; and

-   a pharmaceutically acceptable excipient.

It is yet another aspect of this invention to provide a method ofblocking infection of a respiratory virus in a target cell, comprising:

-   allowing any one of the peptides of this invention to come into    contact with and bind to the respiratory virus in a system    comprising the target cell and the respiratory virus; and-   allowing the peptide to inhibit a late endosome of the target cell    from releasing a viral RNA, thereby blocking the infection of the    respiratory virus in the target cell;-   wherein the respiratory virus is selected from influenza viruses and    coronaviruses; wherein the influenza viruses include influenza virus    subtypes H1, H3, H5, and H7, and the coronaviruses include SARS-CoV    and MERS-CoV.

Preferably, the binding of the peptide to the virus includes the bindingof the peptide to a surface glycoprotein of the virus; and the peptidecan inhibit the viral RNA release by inhibiting pH decrease in the lateendosome.

It is yet another aspect of this invention to provide a method oftherapeutically or preventively treating a subject infected or at therisk of developing infections of respiratory viruses, including a stepof administering to the subject an effective amount of any one of thepeptides of this invention.

It is yet another aspect of this invention to provide a kit forscreening a peptide capable of inhibiting infection of a respiratoryvirus, comprising:

-   a positive control being any one of the peptides of this invention;-   a target cell that can be infected by the respiratory virus;-   an optional cell culture medium; and-   an optional negative control or an optional blank control.

Preferably, the target cell is selected from Madin-Darby canine kidneycell (MDCK, ATCC No. CCL-34), fetal rhesus monkey kidney cell (FRhK-4,ATCC No. CRL-1688) and African green monkey kidney E6 cell (Vero-E6,ATCC No. CRL-1586).

Preferably, the respiratory virus is selected from influenza viruses andcoronaviruses; and wherein the influenza viruses include influenza virussubtypes H1, H3, H5, and H7, and the coronaviruses include SARS-CoV andMERS-CoV.

It is yet another aspect of this invention to provide a method ofscreening a peptide capable of inhibiting infection of a respiratoryvirus, comprising the steps as follows:

-   a) providing an isolated or randomly synthesized candidate peptide;-   b) providing a positive control, the positive control being any one    of the peptides of this invention;-   c) allowing the candidate peptide and the positive control to come    into contact with the respiratory virus, respectively and    separately;-   d) infecting a target cell using the respiratory virus contacted    with the candidate peptide and using the respiratory virus contacted    with the positive control, respectively and separately;-   e) evaluating the capability of the candidate peptide and the    positive control to inhibit infection of the respiratory virus in    the target cell; and-   f) selecting the candidate peptide having an inhibition ability    equal to or superior to that of the positive control.

Advantageous Effects

The present invention provides a peptide capable of suppressing variousrespiratory virus infections and discloses the mechanism of the peptidein inhibition of infections of respiratory viruses. Due to thefunctional domain capable of binding to a surface glycoprotein of arespiratory virus, the peptide can bind to the viral surfaceglycoprotein and in turn be delivered into endosomes of cells viaendocytosis. Since the peptide is rich of basic amino acids, it caninhibit the decrease of pH in late endosomes, thereby blocking thevirus-endosome membrane fusion and subsequent viral disassembly andviral RNA release. Thus, the peptide of the present invention showspotent activity of prevent infections of respiratory viruses such asdiverse subtypes of influenza viruses and coronaviruses, and it can beused for blocking infections of respiratory viruses in target cells andfor prevention or treatment of infections of respiratory viruses. Thepresent invention discloses the mechanism of such a peptide ininhibition of infections of respiratory viruses, which provides atheoretical support for developing new prophylactic and therapeuticagents with broad-spectrum antiviral activities and paves the way fordevelopment of the prophylactic and therapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Preferred embodiments of the present invention will be explained by wayof examples and with reference to the accompanying drawings, in which:

FIGS. 1A-1D show the construction, expression and purification ofrecombinant mouse β-defensin-4 (rmBD4); wherein (A) shows that theoriginal codons of mouse β-defensin-4 (mBD4) gene were optimized to theE. coli-preferred codons by OPTIMIZER; (B) shows that based on theoptimized sequence, six pairs of oligonucleotides were designed forsynthesis of rmBD4 gene by PCR, wherein the underlined nucleotidesindicated recognition sites of KpnI and XhoI; (C) shows SDS-PAGE andWestern blot analysis of the expression and purification of β-defensin-4fusion protein, Trx-rmBD4 (Trx-β-D-4), wherein, in the analysis,proteins in the samples were firstly resolved by 12% (w/v) SDS-PAGE andvisualized by staining with Coomassie brilliant blue solution, parallelresolved proteins on the gel were transferred to PVDF membrane and thendetected by Western blot using Rabbit anti-mBD4 antibody (1:2,000,available from Max Biotechnology Co. Ltd., China) as the primaryantibody and Horseradish Peroxidase conjugated (HRP-) Goat anti-rabbitantibody (1:3,000, available from Dako, Denmark) as the second antibody;and (D) shows tricine gel (16% (w/v)) eletrophoresis analysis ofpurified Trx-β-D-4, digested Trx-β-D-4 and purified rmBD4 (i.e.,Recovered β-D-4 shown in FIG. 1D).

FIGS. 2A-2C show evaluation of antiviral activities and cytotoxicity ofpeptides in vitro; wherein (A) shows screening of antiviral activitiesof 11 short peptides derived from mBD4 in 30 mM phosphate buffer (PB)and MEM by plaque assay of MDCK cell cultures; (B) shows detection ofantiviral efficacy of the P9 (SEQ ID NO: 10) at different concentrationsin PB by plaque assay; and (C) shows detection of cytotoxicity of the P9(SEQ ID NO: 10), smBD4 (SEQ ID NO: 1), and rmBD4 using atetrazolium-based colorimetric (MTT) assay.

FIGS. 3A-3C show evaluation of preventive and therapeutic effects of theP9 (SEQ ID NO: 10) in a lethal virus challenge mouse model; wherein (A)shows survival rate of mice that were intranasally (i.n.) inoculatedwith the P9 (SEQ ID NO: 10) or rmBD4 (50 μg/mouse) before the lethalvirus challenge; (B) shows survival rate of mice that were i.n. treatedwith the P9 (SEQ ID NO: 10) or rmBD4 (50 μg/mouse) at 4 hours after thelethal virus challenge; and (C) shows survival rate of mice that wereinjected intraperitoneally (i.p.) with the P9 (SEQ ID NO: 10) in theindicated dosage (200 or 400 μg/mouse) at 4 hours after the lethal viruschallenge. The statistical significance of survival rates (9 mice/group)was analyzed by GraphPad Prism 5 and the P values were indicated inthese drawings.

FIGS. 4A-4C show detections of viral loads and histopathological changesin lung tissues of the mice preventively and therapeutically treatedwith the P9 (SEQ ID NO: 10) or rmBD4; wherein (A) shows viral RNA copiesin lung tissues of the mice receiving prophylactic treatment(Prevention) or therapy by i.n. inoculation (i.n. therapy) or by i.p.injection (i.p. therapy) measured by real-time RT-PCR, the data beingpresented as mean+SD of results from five mice and the symbol*indicatingP<0.05 as analyzed by two-tailed Student's t-test; (B) shows that thetiters of infectious virus in lung tissues of the mice were alsodetected by plaque assay, the results being presented as mean+SD of fivemice and the symbol*indicating P<0.05 as analyzed by two-tailedStudent's t-test; and (C) provides images showing histopathologicalchanges in the mouse lung tissues tested by H&E staining. Representativehistological sections of the lung tissues taken from the mice treatedwith P9 (SEQ ID NO: 10) (Prevention, i.n. therapy, or i.p. therapy) orwith rmBD4 (Prevention, or i.n. therapy), untreated mice (Untreated) anduninfected mice (Normal) were shown in FIG. 4C (original magnification100×).

FIGS. 5A-5I show that the P9 (SEQ ID NO: 10) inhibited the virusinfection in MDCK cells via reaction with the virus; wherein (A)-(C)show the viral RNA copies inside the infected cells collected at theindicated time-points of post-infection which were detected by real-timeRT-PCR; (D)-(F) show the viral RNA copies in culture supernatantscollected at the indicated time-points of post-infection which weredetected by real-time RT-PCR; and (G)-(I) show virus titers in theculture supernatants collected at the indicated time-points ofpost-infection which were detected by plaque assay. The untreated viruscontrol (VC) was included in the experiments. In these drawings, thedata were presented as mean+SD of three independent experiments and thesymbol*indicated P<0.05 as analyzed by two-tailed Student's t-test.

FIGS. 6A-6C show that the P9 (SEQ ID NO: 10) bound to virus particlesand viral surface protein haemagglutinin (HA); wherein (A) showsrepresentative fluorescence images that were taken by confocalmicroscope (original magnification 400×), the results of which indicatethat P9 (SEQ ID NO: 10) bound to virus but not cells; (B) shows that P9(SEQ ID NO: 10) bound to viral surface glycoprotein HA but notneuraminidase (NA) using Western blot assay; and (C) shows the bindingaffinities of the P9 (SEQ ID NO: 10) to HA and NA detected by ELISA, theresults of which confirm that the P9 (SEQ ID NO: 10) bound to HA but notNA.

FIGS. 7A-7C show that the P9 (SEQ ID NO: 10) did not inhibitvirus-receptor binding and endocytosis but blocked viral RNA releasefrom the late endosomes; wherein (A) and (B) show representativefluorescence images that were taken by confocal microscope (originalmagnification 400×), and more particularly, the results of (A) indicatethat the P9 (SEQ ID NO: 10) could not prevent the virus-receptor bindingand endocytosis, and (B) shows the colocalization (orange spots in colorimage, corresponding to the gray spots in black and white image, as seenin the right picture of (B)) of the virus (green spots in color image,corresponding to the gray spots in black and white image, as seen in theleft picture of (B)) and the P9 (SEQ ID NO: 10) (red spots in colorimage, corresponding to the gray spots in black and white image, as seenin the middle picture of (B)) exhibited after two-hour infection; and(C) shows the results obtained by infecting MDCK cells with theuntreated virus (indicated by “VC”) or with the virus pretreated usingthe P9 (SEQ ID NO: 10) (indicated by “P9”), harvesting the infectedcells at indicated time-points and detecting the viral RNA copies byreal-time RT-PCR, the results being presented as mean±SD of threeindependent experiments.

FIGS. 8A-8B show that the P9 (SEQ ID NO: 10) suppressed pH decrease inlate endosomes to block viral RNA release to the infected cells; wherein(A) shows viral RNA copies in cell samples collected at indicatedtime-points and detected by real-time RT-PCT, the results of whichindicate that the inhibitory effect of the P9 (SEQ ID NO: 10) wassimilar to that of a late endosome inhibitor (NH₄Cl); and (B) providesimages showing the detection of endosomal acidification taken byconfocal microscope, in which the white arrows point to the virallocations (green spots in color image, corresponding to the gray spotsin black and white image, as seen in the lower left picture of (B)) andtheir corresponding endosomal acidification (red spots in color image,corresponding to the gray spots in black and white image, as seen in theupper left picture of (B)), while in the cells infected withP9-pretreated virus, no endosomal acidification (see the upper rightpicture of (B)) but the viruses in endosome (green spots in color image,corresponding to the gray spots in black and white image, as seen in thelower right picture of (B)) were detected.

FIGS. 9A-9C show that basic amino acids in the P9 (SEQ ID NO: 10) play akey role in the inhibition of infections of viruses; wherein (A) showsthe binding affinities of the indicated peptides, including P9-analogouspeptides (see Table 3 below) obtained by substituting 1 to 3 basic aminoacid(s) at C-terminus of the P9 (SEQ ID NO: 10) with neutral or acidicamino acid(s) (P9-S1 (SEQ ID NO:13), P9-S2 (SEQ ID NO:14), and P9-S3(SEQ ID NO:15)), or by adding 3 acidic amino acids at the N-terminus ofthe P9 (P9-aci-1) (SEQ ID NO:16), or by adding 3 basic amino acids atthe N-terminus of the P9 (P9-KHR) (SEQ ID NO:17), to viral protein HAtested at indicated concentrations (ng) by ELISA, with bovine serumalbumin being included as a negative control (NC); (B) shows theinhibitory activities of the peptides against hemagglutination of viralHA protein measured by hemagglutination-inhibition (HAI) assay; and (C)shows the results of detection of antiviral effects of the P9-analogouspeptides in MDCK cells using real-time RT-PCR.

FIGS. 10A-10B show detections of antiviral effects of the P9 (SEQ ID NO:10) against infections of broad respiratory viruses in vitro, whereinthe 50% inhibitory concentration (IC₅₀) was respectively indicated bydotted lines and the results were presented as mean±SD of threeindependent experiments. In FIG. 10, the graph (a) shows detections ofantiviral effects of the P9 (SEQ ID NO: 10) against infections ofdifferent subtypes of influenza A virus by plaque assay; and the graph(b) shows detections of antiviral effects of the P9 (SEQ ID NO: 10)against infections of SARS-CoV and MERS-CoV by plaque assay.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a peptide synthesized through a chemicalroute or by a genetic engineering process. The peptide has a functionaldomain capable of binding to a surface glycoprotein of a respiratoryvirus and has an activity of inhibiting infection of the respiratoryvirus.

For example, in the case that the respiratory virus is an influenzavirus such as influenza virus subtype H1, H3, H5 or H7, the peptide ofthe present invention can bind to the surface glycoprotein HA of thevirus. In the case that the respiratory virus is a coronavirus such asSARS-CoV or MERS-CoV, the peptide of the present invention can bind tothe surface glycoprotein spike protein (S protein) of the virus.

Having the functional domain capable of binding to a surfaceglycoprotein of a respiratory virus, the peptide can bind to the surfaceglycoprotein of the respiratory virus and further can be delivered intoendosomes of cells through endocytosis so as to exert its activity inpreventing infection of the respiratory virus.

Preferably, the peptide of the present invention further has a functionof preventing acidification in late endosomes of cells. In particular,the peptide can suppress pH decrease in late endosomes to block thevirus-endosome membrane fusion and subsequent viral disassembly andviral RNA release.

Preferably, the peptide of the present invention has 5 or more (forexample, less than or equal to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8,7, 6) basic amino acids. Preferably, the peptide has 2 or more (forexample, less than or equal to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3) basicamino acids in N-terminal region or C-terminal region thereof. Morepreferably, the peptide has 3 or more (for example, less than or equalto 12, 11, 10, 9, 8, 7, 6, 5, 4) basic amino acids in N-terminal regionor C-terminal region thereof.

It is also preferred that the peptide of the present invention has 4 ormore (for example, less than or equal to 10, 9, 8, 7, 6, 5), preferably4 to 6, cysteines.

In the present invention, it is preferred that the N-terminal region ofthe peptide comprises a sequence of no more than 10 amino acids countingfrom the N-terminal amino acid of the peptide; and the C-terminal regionof the peptide comprises a sequence of no more than 10 amino acidscounting from the C-terminal amino acid of the peptide.

Preferably, the C-terminal region has two cysteines and is rich of basicamino acids. The expression “rich of basic amino acids” used hereinmeans that the C-terminus of the peptide has 2 or more (for example,less than or equal to 12, 11, 10, 9, 8, 7, 6, 5, 4, 3), preferably 3 to6, basic amino acids.

More preferably, the C-terminal region has 10 amino acids with thefollowing amino acid composition: basic amino acid-neutral aminoacid-basic amino acid-neutral amino acid-basic aminoacid-cysteine-cysteine-basic amino acid-neutral amino acid-basic aminoacid-free carboxyl. As used herein, the “free carboxyl” at theC-terminus refers to the carboxyl of the C-terminal basic amino acid.

It is preferred that the peptide of the present invention has an aminoacid sequence (a) or (b) as described below:

(a) an amino acid sequence as set forth in SEQ ID NO.: 10; or

(b) an amino acid sequence obtained by substitution, deletion and/oraddition of one or several amino acids in the amino acid sequence (a).

The amino acid sequence shown in SEQ ID NO.: 10 comprises the functionaldomain capable of binding to a surface glycoprotein of a respiratoryvirus, and it has two cysteines and is rich of basic amino acids in theC-terminal region thereof. The peptide having an amino acid sequence asset forth in SEQ ID NO.: 10 (i.e., amino acid sequence (a)) is capableof binding to surface glycoproteins of respiratory viruses and has afunction of preventing acidification in late endosomes of cells so as toprevent infections of respiratory viruses.

The peptide having an amino acid sequence (b) described above can beobtained by substitution, deletion and/or addition of one or severalamino acids in the amino acid sequence (a), as long as the substitution,deletion and/or addition do/does not substantively affect the activityof the peptide in preventing infections of respiratory viruses.Preferably, the number of the basic amino acids remains unchanged orincreases in the amino acid sequence obtained by substitution, deletionand/or addition of one or several amino acids. It is preferred that theamino acid sequence (b) is at least 70%, preferably at least 80%, morepreferably at least 90%, and still more preferably at least 97%,identical to the amino acid sequence (a).

It is also preferred that the amino acid sequence of the peptideaccording to the present invention is as set forth in SEQ ID NO.: 10,SEQ ID NO.: 13, or SEQ ID NO.: 17. As described above, the peptidehaving an amino acid sequence as set forth in SEQ ID NO.: 10 can bind tosurface glycoproteins of respiratory viruses and has a function ofpreventing acidification in late endosomes of cells so as to preventinfections of respiratory viruses. The peptide having an amino acidsequence as set forth in SEQ ID NO.: 13 was obtained by substituting onebasic amino acid K with N at the C-terminus of the amino acid sequenceshown in SEQ ID NO.: 10. It has been demonstrated that such substitutiondoes not affect the binding affinities of the peptide to surfaceglycoproteins of respiratory viruses and does not substantivelyinfluence the effect of the peptide against infections of respiratoryviruses. The peptide as set forth in SEQ ID NO.: 17 was obtained byadding 3 basic amino acids K, H, and R at the N-terminus of the aminoacid sequence shown in SEQ ID NO.: 10. It has been demonstrated thatsuch addition does not affect the binding affinities of the peptide tosurface glycoproteins of respiratory viruses, nor does it influence theeffect of the peptide against infections of respiratory viruses.

Preferably, the peptide of the present invention is obtained by geneticengineering (for example, obtained as a recombinant peptide) or bychemical synthesis methods. In addition, it is preferred that thepeptide of the present invention is originated from mouse or human, andmore preferably, the peptide is originated from mouse β-defensin-4(mBD4).

Another aspect of the disclosure provides a composition comprising oneor more peptides according to the present invention and apharmaceutically acceptable excipient.

The amount of the peptide used in the composition can be determineddepending on specific applications and may be in the range of 5-500μg/ml, preferably of 20-300 μg/ml and more preferably of 50-200 μg/ml.

The pharmaceutically acceptable excipients useful in the presentinvention are those conventionally used in the art. The excipients canbe appropriately selected according to the requirements of practicalapplications or specific dosages, and may be those disclosed, forexample, in Chapter 4, “Research of Peptide Drug Formulations”, pages82-100 of “Peptide Drugs Research and Development” edited by Baoqiu L I,People's Medical Publishing House, July 2011. In the composition of thepresent invention, the pharmaceutically acceptable excipient may be usedin an amount within a common range, and the suitable amount thereof canbe determined by those of ordinary skill in the art according topractical applications.

Another aspect of the disclosure provides a method of blocking infectionof a respiratory virus in a target cell, comprising:

-   allowing a peptide according to the present invention to come into    contact with and bind to the respiratory virus in a system    comprising the target cell and the virus; and-   allowing the peptide to inhibit a late endosome of the target cell    from releasing a viral RNA, thereby blocking infection of the    respiratory virus in the target cell.

Preferably, the respiratory virus is selected from influenza viruses andcoronaviruses. More preferably, the influenza viruses include influenzavirus subtypes H1, H3, H5, and H7, and the coronaviruses includeSARS-CoV and MERS-CoV.

Preferably, the binding of the peptide to a virus includes binding ofthe peptide to a surface glycoprotein of the virus. Further, the peptidecan inhibit the viral RNA release by inhibiting pH decrease in the lateendosome. In this way, the peptide exerts the effect of blockinginfections of respiratory viruses in target cells.

In one specific embodiment of the present invention, the method ofblocking infection of a respiratory virus in a target cell is carriedout in vivo or in vitro.

Another aspect of the disclosure provides use of the peptide of thepresent invention for the manufacturing of a medicament for preventionor treatment of infections of respiratory viruses. The medicament may beprovided in a spray form, an injection form (such as an injectionliquid, or a freeze-dry powder injection), an oral formulation or thelike.

Another aspect of the disclosure provides a method of therapeutically orpreventively treating a subject infected or at the risk of developinginfections of respiratory viruses, including the step of administeringto the subject an effective amount of any one or more of the peptidesaccording to the present invention.

In the case that the peptide is used for treating a subject infected bya respiratory virus, the administration dose is an effective amount fortherapy. For example, the dose of the total of the peptides of thepresent invention used in the therapy may be in the range of 1-40 mg/kgweight/day, preferably 2-20 mg/kg weight/day, and more preferably 2.5-10mg/kg weight/day.

In the case that the peptide is used for prophylactic treatment of asubject at the risk of developing infections of respiratory viruses, theadministration dose is an effective amount for prevention. For example,the dose of the total of the peptides of the present invention used inthe prophylactic treatment may be in the range 0.1-40 mg/kg weight/day,preferably of 0.5-20 mg/kg weight/day, and more preferably 1-10 mg/kgweight/day.

The peptide of the present invention may be administrated by intranasalinoculation (for example, spray) or by intravenous injection (forexample, intraperitoneal injection).

The respiratory viruses described herein preferably include influenzaviruses and coronaviruses. It is more preferred that the influenzaviruses include influenza virus subtypes H1, H3, H5 and H7, such asH1N1, H3N2, H5N1, H7N7 and H7N9, and the coronaviruses include SARS-CoVand MERS-CoV.

Another aspect of the disclosure provides a method of preparing thepeptide according to the present invention, comprising synthesizing thepeptide through a chemical route or by a biologic method (i.e., geneticengineering).

The peptide as desired may be synthesized by a chemical method or abiologic method through a general process in the art. For example, bylinking the constitutive amino acids one by one via chemical reactions,the desired peptide can be synthesized chemically. Synthesis of thedesired peptide by a biologic method may include, for example, thefollowing steps: amplifying the DNA encoding the desired peptide viapolymerase chain reaction (PCR); subcloning the DNA into an expressionvector; and transfecting or transforming the expression vectorcontaining the DNA into eukaryotic or prokaryotic host cells, therebyexpressing the desired peptide.

Another aspect of the disclosure provides an isolated DNA encoding anyone of the peptides according to the present invention.

Another aspect of the disclosure provides an expression vectorcontaining the DNA according to the present invention operatively linkedto a promoter.

As used herein, the term “expression vector” refers to a vector capableof directing the expression of the gene to which it is operativelylinked. The expression of the corresponding peptide according to thepresent invention may be directed by a promoter sequence, by operativelylinking the promoter sequence to the DNA of the invention to beexpressed. In general, expression vectors useful in genetic engineeringtechniques are often in the form of plasmids. However, the disclosure isintended to include other known forms of expression vectors.

A promoter and a DNA encoding the peptide of the invention are“operatively linked” when the promoter is capable of driving expressionof the DNA into RNA. Said promoter may be any promoter conventionallyused in the field of genetic engineering.

The expression vector of the invention may also contain additionalsequence or sequences, such as termination sequence which can serve toenhance message levels and to minimize readthrough from the constructinto other sequences. In addition, the expression vector may furtherhave selectable markers, for example, in the form of antibioticresistance genes, which permit screening out those cells carrying thesevectors.

Another aspect of the disclosure provides a host cell containing theexpression vector according to the present invention.

The term “host cell” used herein refers to a cell into which theexpression vector according to the present invention has beenintroduced. Such cells may be prokaryotic, which can be used, forexample, to rapidly produce a large amount of the expression vectors ofthe invention.

The host cells can be transiently or stably transformed using theexpression vectors of the invention. Such transform of expressionvectors into cells can be accomplished via any technique known in theart, including but not limited to standard bacterial transformations,calcium phosphate co-precipitation or electroporation.

Another aspect of the disclosure provides a kit for screening a peptidecapable of inhibiting infection of a respiratory virus, comprising: apositive control, the control being any one of the peptides according tothe present invention; and a target cell that can be infected by therespiratory virus.

Preferably, the kit may further comprise a cell culture medium. Morepreferably, the kit may further comprise a negative control or a blankcontrol. The negative control or blank control is a peptide that cannotinhibit infection of the respiratory virus in the target cell.

Said target cell is the one to be infected by the respiratory virus andmay be selected from Madin-Darby canine kidney cell (MDCK, ATCC No.CCL-34), fetal rhesus monkey kidney cell (FRhK-4, ATCC No. CRL-1688) andAfrican green monkey kidney E6 cell (Vero-E6, ATCC No. CRL-1586). Therespiratory virus may be selected from influenza viruses andcoronaviruses. Preferably, the influenza viruses include influenza virussubtypes H1, H3, H5, and H7, and the coronaviruses include SARS-CoV andMERS-CoV.

Another aspect of the disclosure provides a method of screening apeptide capable of inhibiting infection of a respiratory virus,comprising the steps as follows:

a) providing an isolated or randomly synthesized candidate peptide;

b) providing a positive control, the positive control being any one ofthe peptides of the invention;

c) allowing the candidate peptide and the positive control to come intocontact with the respiratory virus, respectively and separately;

d) infecting a target cell using the respiratory virus contacted withthe candidate peptide and using the respiratory virus contacted with thepositive control, respectively and separately;

e) evaluating the capability of the candidate peptide and the positivecontrol to inhibit infection of the respiratory virus in the targetcell; and

f) selecting the candidate peptide having an inhibition ability equal toor superior to that of the positive control.

In a specific embodiment of the present invention, the method ofscreening a peptide capable of inhibiting infection of a respiratoryvirus is carried out in vivo or in vitro.

Hereinafter, the present invention will be described in more details byway of examples with reference to the figures. The objects, features,and aspects of the present invention are disclosed in or are apparentfrom the following description. It is to be understood by one ofordinary skill in the art that the description is provided for thepurpose of illustrating exemplary embodiments only, and is not intendedto limit broader aspects of the present invention, which broader aspectsare embodied in the exemplary constructions.

Materials and Methods

Viruses and Cell Culture

A highly virulent mouse adapted mutant strain of influenza A virusA/Hong Kong/415742Md/2009 (H1N1) (see “Zheng, B., et al. D225G mutationin hemagglutinin of pandemic influenza H1N1 (2009) virus enhancesvirulence in mice. Exp Biol Med (Maywood) 235, 981-988 (2010)”), A/HongKong/8/68 (H3N2) (ATCC No. VR-544), A/Vietnam/1194/2004 (H5N1) (see“Chan M C, et al. Proinflammatory cytokine responses induced byinfluenza A (H5N1) viruses in primary human alveolar and bronchialepithelial cells. Respir Res. Nov. 11, 2005; 6:135”),A/Netherlands/219/2003 (H7N7) (see “Fouchier R A, et al. Avian influenzaA virus (H7N7) associated with human conjunctivitis and a fatal case ofacute respiratory distress syndrome. Proc. Natl. Acad. Sci. USA101:1356-1361. 2004”) and A/Anhui/1/2013 (H7N9) (see “Gao R, et al.Human infection with a novel avian-origin influenza A (H7N9) virus. NEngl J Med. 368(20):1888-97. May 2013”) were cultured in Madin-Darbycanine kidney (MDCK, ATCC No. CCL-34) cells and their titers weredetermined by plaque and TCID₅₀ assays (see “Zheng, B. J., et al.Delayed antiviral plus immunomodulator treatment still reduces mortalityin mice infected by high inoculum of influenza A/H5N1 virus. Proc NatlAcad Sci USA 105, 8091-8096 (2008)”). Severe acute respiratory syndromecoronavirus (SRAS-CoV) strain HKU39849 was cultured in fetal rhesusmonkey kidney (FRhK-4, ATCC No. CRL-1688) cells (see “Peiris J S, et al.Coronavirus as a possible cause of severe acute respiratory syndrome.Lancet. 361(9366):1319-25. April 2003”), and Middle East respiratorysyndrome coronavirus (MERS-CoV) strain hCoV-EMC/2012 (provided by Dr.Ron Fouchier, Erasmus University Medical Center Rotterdam) was culturedin African green monkey kidney E6 (Vero-E6, ATCC No. CRL-1586) cells,and their titers were determined by plaque and TCID₅₀ assays (see“Zhong, N. S., et al. Epidemiology and cause of severe acute respiratorysyndrome (SARS) in Guangdong, People's Republic of China, in February,2003. Lancet 362, 1353-1358 (2003)”).

Cloning, Expression and Purification of Recombination Mouse Defensin-4

The codons of mouse β-defensin-4 (mBD4) were optimized to E.coli-preferred codons based on OPTIMIZER (FIG. 1A) (see “Puigbo, P.,Guzman, E., Romeu, A. & Garcia-Vallve, S. OPTIMIZER: a web server foroptimizing the codon usage of DNA sequences. Nucleic Acids Res 35,W126-131 (2007)”). The gene fragment of mBD4 was generated by PCR-basedgene synthesis using 6 pairs of oligonucleotides (FIG. 1B). Thecodon-optimized gene was cloned into PCR2.1 vector (Invitrogen, USA) andthen sub-cloned into PET32a(+) (Novagen, USA) at KpnI and XhoI sites.The resulting plasmid was transformed into BL21 (DE3) to expressthioredoxin-β-defensin-4 fusion protein (Trx-β-D-4). Trx-β-D-4 wasreleased from the E. coli cytoplasm by a simple osmotic shock procedure(see “McCoy, J. & Lavallie, E. Expression and purification ofthioredoxin fusion proteins. Curr Protoc Mol Biol Chapter 16, Unit 16 18(2001)”) and was further purified by AKTA-FPLC (GE Healthcare, UnitedKingdom) using His Trap FF column according to the manufacturer'sinstruction (FIG. 1C). 80 μg of Trx-β-D-4 was digested with 1Uenterokinase (Merck, USA) to release recombination mouse β-defensin-4(rmBD4, or β-D-4 for short). β-D-4 was recovered by cation-exchangechromatography using Sp sepharose Fast Flow (GE Healthcare) according tothe manufacturer's instruction. Purified β-D-4 was desalted using PD-10column (GE Healthcare) into 25 mM HEPES buffer (pH 7.4) (FIG. 1D).

Peptide Design and Evaluation of Antiviral Effects

Full length mBD4 (smBD4) (SEQ ID NO: 1) and short peptides derived frommBD4 were designed as shown in Table 1, and they were chemicallysynthesized and confirmed by Mocell Biotech Limited (Shanghai, China).Antiviral effects of the short peptides, smBD4 (SEQ ID NO: 1), and rmBD4were initially evaluated in a low-salt medium, i.e., 30 mM phosphatebuffer (PB) containing 24.6 mM Na₂HPO₄ and 5.6 mM KH₂PO₄, pH 7.4 (see“Gong, T., et al. Recombinant mouse beta-defensin 2 inhibits infectionby influenza A virus by blocking its entry. Arch Virol 155, 491-498(2010)”), and in a high-salt medium MEM (Invitrogen, USA). The peptides(25 μg/ml) were premixed with H1N1 virus (50 PFU/well) in PB or MEM andincubated at room temperature (RT) for 1 hour. MDCK cells were infectedby the peptide-pretreated virus and antiviral activities of the peptideswere measured using a plaque assay (see “Sui, H. Y., et al. Smallinterfering RNA targeting m2 gene induces effective and long terminhibition of influenza A virus replication. PLoS One 4, e5671 (2009)”).

TABLE 1Sequences of smBD4 (SEQ ID NO: 1) and short peptides derived from mBD4Peptide name Sequence SEQ ID No. smBD4iinnpitcmtngaicwgpcptafrqigncghfkvrcckir 1 P1 iinnpitcmt 2 P2     itcmtngaic 3 P3           ngaicwgpcp 4 P4                wgpcptafrq5 P5                     tafrqigncg 6 P6                         igncghfkvr 7 P7                              hfkvrcckir 8 P8                    tafrqigncghfkvrcckir 9 P9          ngaicwgpcptafrqigncghfkvrcckir 10 P10 iinnpitcmtngaicwgpc 11P11 iinnpitcmtngaicwgpcptafrqigncg 12Cytotoxicity and IC₅₀ Assays

Cytotoxicity of the peptides was determined by detection of 50% toxicconcentration (TC₅₀) using a tetrazolium-based colorimetric (MTT) assay(see “Pauwels, R., et al. Rapid and automated tetrazolium-basedcolorimetric assay for the detection of anti-HIV compounds. J VirolMethods 20, 309-321 (1988)”). Briefly, MDCK cells were cultured in a96-well cell culture plate overnight. The cells were washed twice withPBS (Invitrogen, USA) (the PBS described hereinafter was also fromInvitrogen, USA), then added with 100 μl/well of MEM containing variousconcentrations of peptides or without peptides in triplicates. Afterincubated at 37° C. for 24 hours, 10 μl/well of MTT solution (Beyotime,China) was added to the plate. After the plate was incubated for 4hours, 100 μl of 10% (w/v) SDS in 0.01M HCl was added to each well.After further incubation at 37° C. overnight, the plate was read atOD₅₇₀ and OD₆₄₀ using Victor™ X3 Multilabel Reader (PerkinElmer, USA).

Experiments were performed according to the methods disclosed in thereference below, except that 50% inhibitory concentration (IC₅₀) of thepeptides against infections of different subtypes of influenza A virus,i.e., H1N1, H3N2, H5N1, H7N7 and H7N9, coronaviruses SARS-CoV andhCoV-EMC were determined directly by plaque assay, and/or virus titersin culture supernatants collected at 18 hours post-infection weredetected indirectly using plaque assay and real-time RT-PCR (see “Zheng,B. J., et al. Delayed antiviral plus immunomodulator treatment stillreduces mortality in mice infected by high inoculum of influenza A/H5N1virus. Proc Natl Acad Sci USA 105, 8091-8096 (2008)”).

Evaluation of Antiviral Effects In Vivo

BALB/c female mice (17-21 g), 6-8 weeks old, were kept in biosafetylevel 3 laboratory and given access to standard pellet feed and water.All experimental protocols followed the standard operating procedures ofthe approved biosafety level 3 animal facilities and were approved bythe Animal Ethics Committee (see “Zheng, B. J., et al. Delayed antiviralplus immunomodulator treatment still reduces mortality in mice infectedby high inoculum of influenza A/H5N1 virus. Proc Natl Acad Sci USA 105,8091-8096 (2008)”). The highly virulent mouse adapted mutant strainA/Hong Kong/415742Md/2009 (H1N1) was used for lethal challenge of themice. To evaluate prophylactic effects, the mice were intranasally(i.n.) inoculated with 50 μg/mouse of the P9 (SEQ ID NO: 10) or rmBD4and then challenged with 5 LD₅₀ of the virus. For evaluation oftherapeutic effects, the mice were challenged with 5 LD₅₀ of the virusand i.n. inoculated with 3 doses of the P9 (SEQ ID NO: 10) or rmBD4 (50μg/mouse/day) at 1 day interval, or intraperitoneally (i.p.) injectedwith 200 or 400 μg/mouse/day of the P9 (SEQ ID NO: 10) for 6 days at 1day interval, which started 4 hours after the lethal challenge. Survivaland general conditions were monitored for 21 days or until death. Forvirological and pathological tests, mice were sacrificed five days afterthe challenge. Blood and lung samples were collected.

Histopathological Staining

Lung tissues collected from the challenged mice were immediately fixedin 10% (v/v) formalin in PBS buffer, applied to dehydration and embeddedin paraffin wax. Sections of 4-6 μm thickness were mounted on slides.Histopathological changes were examined by hematoxylin and eosin (H&E)staining under a light microscope (see “Zheng, B. J., et al. Delayedantiviral plus immunomodulator treatment still reduces mortality in miceinfected by high inoculum of influenza A/H5N1 virus. Proc Natl Acad SciUSA 105, 8091-8096 (2008)”).

Viral RNA Extraction and Real-Time RT-PCR

Viral loads were detected by real-time RT-PCR (see “Zheng, B., et al.D225G mutation in hemagglutinin of pandemic influenza H1N1 (2009) virusenhances virulence in mice. Exp Biol Med (Maywood) 235, 981-988(2010)”). Briefly, viral RNA was extracted from culture supernatantsusing RNeasy Mini Kit (Qiagen, USA) according to the manufacturer'sprotocols, while viral RNA in cell lysis and mouse lung tissues wasextracted using QIAamp Viral RNA Mini Kit (Qiagen) according to themanufacturer's protocols. Reverse transcription was performed using RSIIkit (Invitrogen, USA) according to the manufacture's instruction.Real-time PCR was performed using ABI SYBR Green Mastermix and the 7500system (Invitrogen). Clones of HA gene of H1N1 virus were used as thepositive control and standard. Real-time PCR experiments were performedin triplicate.

Fluorescence Image Assay

The H1N1 virus was labeled with green fluorescent lipophilic dye Dio(Invitrogen, USA) according to the manufacture's instruction, and the P9(SEQ ID NO: 10) was red labeled with 1:5000 diluted Rabbit-anti-smBD4antibody (Max Biotechnology) and 1:400 diluted Goat anti-rabbit Alexa594antibody (Invitrogen, USA) so as to detect the P9 (SEQ ID NO: 10). Thecell membrane was stained with dye Alexa594 (Invitrogen) according tothe manufacture's instruction. The labeled virus and P9 (SEQ ID NO: 10)were premixed for 1 hour and then incubated with MDCK cells at 4° C. or37° C. The infected cells were fixed by 10% (v/v) formalin in PBS bufferat different time points of post-infection and images were taken byconfocal microscopy (Carl Zeiss LSM 700, Zeiss, Germany).

Detection of the Endosomal Acidification

The ensosomal acidification after the viral infection was detectedaccording to the manufacturer's instruction of pH-sensitive dye, i.e.,pHrodo Red dextran (Invitrogen, USA). Briefly, H1N1 virus waspre-labeled with Dio and then incubated with 50 μg/ml of the P9 (SEQ IDNO: 10) (hereinafter referred to as “P9 treated”) or PB (hereinafterreferred to as “untreated”) at room temperature for 45 min, followed byincubation at 4° C. for 15 min. MDCK cells were inoculated with 5 MOI ofthe P9-treated or untreated virus and incubated at 4° C. for 1 hour. 100μg/ml of pH-sensitive dye, i.e., pHrodo Red dextran, was added to thecells and the incubation was continued at 4° C. for 10 min, and then thecells were further cultured at 37° C. for 10 min. After the cells werewashed two times with PBS, fresh media was added thereto and images weretaken immediately by confocal microscopy (Carl Zeiss LSM 700, Zeiss,Germany).

Western Blot Assay

Viral protein bound by the peptide was identified by Western blot assay(see “Guan, Y., et al. Isolation and characterization of viruses relatedto the SARS coronavirus from animals in southern China. Science 302,276-278 (2003)”). Briefly, viral protein samples were fractionated bySDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membrane(Hi-bond Amersham Biosciences, USA). Signal Boost ImmunoreactionEnhancer Kit (EMD MILLPORE, Germany) was used to dilute the primaryantibodies and second antibodies. After the viral protein was bound withindicated antibodies or peptide, immunoreactive bands were visualized byenhanced chemiluminescence.

ELISA

Binding affinities of the peptides to viral protein were detected byELISA (see “Du, L., et al. Intranasal vaccination of recombinantadeno-associated virus encoding receptor-binding domain of severe acuterespiratory syndrome coronavirus (SARS-CoV) spike protein induces strongmucosal immune responses and provides long-term protection againstSARS-CoV infection. J Immunol 180, 948-956 (2008)”). Briefly, differentconcentrations of peptides were coated to the ELISA plates and incubatedwith blocking buffer (5% (w/v) bovine serum albumin in PBS) at 4° C.overnight. After H1N1 viral HA or NA (Invitrogen, USA) was added andincubated at 37° C. for 2 hours, the binding affinities of the peptidesto viral protein were determined by Rabbit anti-His (1:2,000, Santa CrviBiotechnology, USA) or Rabbit anti-HA or Rabbit anti-NA antibodies(1:2,000, Immune Technology, USA) and recognized by Goat anti-rabbitIgG-HRP as the second antibody (1:2,000, Invitrogen, USA) and read in anELISA reader (Victor 1420 Multilabel Counter; PerkinElmer, USA).

Statistical Analysis

Survival of mice and the statistical significance were analyzed byGraphPad Prism 5 (http://www.graphpad.com/scientific-software/prism/).Statistical significance of the other results was calculated by thetwo-tailed Student t test using Stata statistical software. Results wereconsidered significant at P<0.05.

EXAMPLES Experimental Example 1: The Antiviral Peptide P9 (SEQ ID NO:10) Exhibited the Highest Antiviral Activity in Cultured Cells

Eleven mBD4-derived peptides were designed and synthesized (Table 1).Antiviral effects of smBD4 (SEQ ID NO: 1), rmBD4 and 11 peptides derivedfrom mBD4 (Table 1) were detected against the infection of influenza Avirus (H1N1) in cell cultures. Compared to the full-length syntheticmBD4 (smBD4) (SEQ ID NO: 1) and recombinant mBD4 (rmBD4), a shortpeptide P9 (30 amino acids) (SEQ ID NO: 10) exhibited the potent anddose-dependent antiviral activity, while the other short peptides showedmedian or no antiviral effects (FIG. 2). In FIG. 2A, H1N1 virus waspre-incubated respectively with the short peptides and positive controls(i.e., smBD4 (SEQ ID NO: 1) and rmBD4) at room temperature (RT) for 1hour, and then was inoculated to MDCK cells. The inhibitory effects tothe viral infection were detected by plaque assay. The infection ratiowas calculated as the plaque number of virus pretreated with a peptidebeing divided by the plaque number of virus pretreated with PB. Datawere presented as mean+SD of three independent experiments. As shown inFIG. 2A, the P9 (SEQ ID NO: 10) showed the highest antiviral effect, andits shorter forms P7 (SEQ ID NO: 8) and P8 (SEQ ID NO: 9) showed medianantiviral effects. In addition, P9 (SEQ ID NO: 10), smBD4 (SEQ ID NO:1), and rmBD4 were more effective in low-salt phosphate buffer (PB) thanin high-salt minimum essential medium (MEM). In FIG. 2B, after the H1N1virus (50 PFU/well) was pretreated with different concentrations of theP9 (SEQ ID NO: 10), smBD4 (SEQ ID NO: 1), or rmBD4, it was inoculated toMDCK cells. The antiviral activities were determined directly by plaqueassay. The IC₅₀ was indicated by dotted lines and the results showedthat the mean IC₅₀s of smBD4 (SEQ ID NO: 1), rmBD4 and the P9 (SEQ IDNO: 10) were 3.2 μg/ml (0.7 μM), 1.5 μg/ml (0.33 μM) and 1.2 μg/ml (0.36μM), respectively. Data were presented as mean±SD of three independentexperiments. The IC₅₀ of P9 (SEQ ID NO: 10) was about 1.2 μg/ml, whichwas lower than that of smBD4 (SEQ ID NO: 1) or rmBD4 (about 3.2 and 1.5μg/ml, respectively) (FIG. 2B). Furthermore, in FIG. 2C, 50% toxicconcentrations (TC₅₀) of the P9 (SEQ ID NO: 10), smBD4 (SEQ ID NO: 1),and rmBD4 were determined using a tetrazolium-based colorimetric (MTT)assay. The results were presented as optical density (OD) of treatedcells/OD of untreated normal cells (i.e., OD ratio) and the TC₅₀ wasindicated by dotted lines. The data were presented as mean±SD of threeindependent experiments. The results showed that TC₅₀s of the P9 (SEQ IDNO: 10), smBD4 (SEQ ID NO: 1), and rmBD4 were 860, 94 and 580 μg/ml,respectively (Table 2 and FIG. 2C). The cytotoxicity of the P9 (SEQ IDNO: 10) was lowest, which was 9 folds lower than that of smBD4 (SEQ IDNO: 1). Considering the high cytotoxicity of smBD4 (SEQ ID NO: 1), theinventors did not include it in the animal experiments. The selectivityindex (TC₅₀/IC₅₀) of the P9 (SEQ ID NO: 10) was 717, which was about 25and 2 folds higher than that of smBD4 (SEQ ID NO: 1) (selectivity index:29) and rmBD4 (selectivity index: 387), respectively. These resultsdemonstrated that the P9 (SEQ ID NO: 10) exhibited the highest antiviralactivity and selectivity index in vitro.

TABLE 2 Detection of cytotoxicity of smBD4 (SEQ ID NO: 1), rmBD4 and theP9 (SEQ ID NO: 10) Peptide OD of tested cells/OD of normal cells (μg/ml)P9 smBD4 rmBD4 1000 0.386618 — — 750 0.597793 — 0.356453 500 0.833553 —0.550955 250 0.95272 0.365094 0.676465 125 0.946735 0.454871 0.8721462.5 0.960327 0.600363 0.903614 31.25 1.08 0.718644 0.940813 15.60.95876 0.852515 1.03708 7.8 1.023 0.93702 1.056 TC₅₀ (μg/ml) 860 94 580

Experimental Example 2: The P9 (SEQ ID NO: 10) Showed Much PotentProtective Effects Against Lethal Challenge of H1N1 Influenza Virus

Prophylactic and therapeutic effects of the P9 (SEQ ID NO: 10) wereevaluated in lethal infection animal model of H1N1 influenza virus (FIG.3). When the mice were i.n. inoculated with the P9 (SEQ ID NO: 10) orrmBD4 before they were i.n. inoculated with lethal dose of the virus,the survival rate of the P9-treated mice was 100% (9/9), which wassignificantly higher than that of rmBD4-treated mice (22% (2/9))(P<0.008) and that of untreated mice (0%) (P<0.0001) (FIG. 3A). Toevaluate therapeutic effect of the P9 (SEQ ID NO: 10), the mice werei.n. inoculated with 3 doses (50 μg/dose/day) of the P9 (SEQ ID NO: 10)or rmBD4 at four hours after the lethal challenge at one day interval(FIG. 3B). The results showed that the survival rates of mice i.n.treated with the P9 (SEQ ID NO: 10) and rmBD4 were 67% (6/9) and 33%(3/9), respectively. The survival rate of i.n. P9-treated mice wassignificantly higher than that of untreated control (P<0.015) andnumerically higher than that of rmBD4-treated mice (FIG. 3B).Furthermore, to evaluate the effect of administration does, the micewere i.p. given 6 doses (200 or 400 μg/dose/day) of the P9 (SEQ ID NO:10) at one day interval which started four hours post-challenge. Theresults showed that the survival rate was 56% (5/9) when the mice werei.p. injected with 400 μg/dose/day of the P9 (SEQ ID NO: 10), which wassignificantly higher than that of untreated mice (P<0.026), whereas thesurvival rate reduced to 22% (2/9) when the mice were i.p. given 200μg/dose/day of the P9 (FIG. 3C). These results exhibited dose-dependenteffect. In this study, i.n. rmBD4-pretreated or -treated mice showedinferior efficacy compared to the P9 (SEQ ID NO: 10), andrmBD4-pretreated or -treated mice did not show statistically significantincrease of survival rate (P>0.05).

The virus RNA copies and viral titers in lung tissues of mice receivingprophylactic treatment, i.n. therapy or i.p. therapy were detected byreal-time RT-PCR (FIG. 4A) and plaque assay (FIG. 4B), respectively. Theresults showed that the viral loads in lung tissues of P9-pretreated andi.n. or i.p. P9-treated mice were significantly lower than those of theuntreated mice (P<0.05). Histopathological changes in lung tissues wereexamined by H&E staining, which showed that the alveolar damage andinterstitial inflammatory infiltration in the mice pretreated or treatedwith the P9 (SEQ ID NO: 10) were much less severe than those pretreatedor treated by rmBD4 and the untreated mice (FIG. 4C). The alveolardamage and interstitial inflammatory infiltration in the mice treatedwith 400 μg/dose/day of the P9 (SEQ ID NO: 10) were also less severethan those in the mice treated with 200 μg/dose/day of the P9 (SEQ IDNO: 10) (FIG. 4C). The histopathological results were consistent withthose of survivals and viral loads of lung tissues. These resultsindicated that the P9 (SEQ ID NO: 10) has much more potent prophylacticand therapeutic effects against lethal infection of the H1N1 virus thanrmBD4 in vivo.

Experimental Example 3: The P9 (SEQ ID NO: 10) Inhibited Influenza VirusInfection Through Binding to Viral Surface Glycoprotein HA

To investigate how the P9 (SEQ ID NO: 10) inhibited the virus infection,the P9 (SEQ ID NO: 10) was used for pretreating the MDCK cells or H1N1virus before viral infection, or was just supplemented in the cellculture medium after the viral infection. In FIGS. 5A, 5D and 5G, MDCKcells were infected with the virus at MOI of 0.3 and then cultured inthe presence of the P9 (SEQ ID NO: 10) (25 μg/ml) (indicated byP9-maint). In FIGS. 5B, 5E and 5H, the cells were pretreated with the P9(SEQ ID NO: 10) (25 μg/ml) for 1 hour and then infected with 0.3 MOI ofthe virus (indicated by P9-cell-pre). In FIGS. 5C, 5F and 5I, the virus(0.3 MOI) was pretreated with the P9 (25 μg/ml) (SEQ ID NO: 10) for 1hour and then inoculated to the cells (indicated by P9-virus-pre). Theviral loads inside the infected cells (FIGS. 5A, 5B and 5C) and in cellculture supernatants (FIGS. 5D, 5E and 5F) were determined by real-timeRT-PCR, while the titers of infectious virus in supernatants weredetected by plaque assay (FIGS. 5G, 5H and 5I), at different time-pointspost-infection. The results showed that the P9 (SEQ ID NO: 10) had nosignificant inhibitory effects against the virus replication and releasewhen it was maintained in the culture media (FIGS. 5A, 5D and 5G). Thevirus infection was also not inhibited when the cells were pretreatedwith the P9 (SEQ ID NO: 10) (FIGS. 5B, 5E and 5H). Nevertheless, theviral loads both inside the cells and in culture supernatants weresignificantly reduced when the virus was pretreated with the P9 (SEQ IDNO: 10) (P<0.05, FIGS. 5C, 5F and 5I). These results indicated that (1)the P9 (SEQ ID NO: 10) was unable to inhibit the virus replication inthe cells and release from the cells because the viral loads eitherinside the infected cells or in culture supernatants in the presence ofthe P9 (SEQ ID NO: 10) during the culture period were similar to that ofthe untreated virus control (indicated by VC); and (2) the P9 (SEQ IDNO: 10) could inhibit the virus infection by binding to the virus, butnot the surface of target cells, to inhibit the virus infection.

To confirm that the P9 (SEQ ID NO: 10) inhibited the virus infection bybinding to the virus surface, the inventors labeled the virus with greenfluorescence and labeled the P9 (SEQ ID NO: 10) with red fluorescence toview their interaction. The Dio dye labeled virus (5 MOI) was pretreatedwith 50 μg/ml of the P9 (SEQ ID NO: 10) (indicated by P9-Virus-Pre inFIG. 6A) or PB (indicated by VC in FIG. 6A) at RT for 1 hour, or MDCKcells were pretreated with 50 μg/ml of the P9 (SEQ ID NO: 10) (indicatedby P9-Cells-Pre in FIG. 6A) at RT for 1 hour. The MDCK cells wereinfected with the virus (5 MOI) and incubated at 37° C. for 1 hour.Then, the cells were fixed by 10% (v/v) formalin in PBS buffer andstained with rabbit-anti-smBD4 antibody and Alex594 labeledgoat-anti-rabbit antibody. Finally, the cell nucleus was stained withProlong Gold Antifade Reagent with DAPI (Invitrogen, USA).Representative images were taken by confocal microscope (originalmagnification 400×). As shown in FIG. 6A, the red-labeled P9 (SEQ ID NO:10) (corresponding to the gray spots in black and white image in themiddle picture in the first row) bound to the green-labeled virusparticles (corresponding to the gray spots in black and white image inthe left picture in the first row) when the virus was pretreated withthe P9 (SEQ ID NO: 10), and it was carried into the cells (orange spotsin color image, corresponding to the gray spots in black and whiteimage, as seen in the right picture in the first row), whereas no P9(SEQ ID NO: 10) was detected on the cell membrane or inside the cellswhen the cells were pretreated with the labeled P9 (SEQ ID NO: 10). Theresults confirmed that the P9 (SEQ ID NO: 10) inhibited the viralinfection by binding to the viral particle.

The inventors further identified which viral surface protein was boundby the P9 (SEQ ID NO: 10) (FIGS. 6B and 6C). In FIG. 6B, viral proteinsof H1N1 virus were separated by 10% (w/v) SDS-PAGE and transferred toPVDF membrane. Lane 1 represents the viral proteins stained by Coomassiebrilliant blue solution. As for lane 2, the transferred membrane wasincubated with the P9 (SEQ ID NO: 10) (50 μg/ml) for 1 hour and thenwith rabbit-anti-smBD4 antibody (1:2000, Max Biotechnology) for another1 hour for detection of the P9 (SEQ ID NO: 10) binding. As for lane 3,the membrane was incubated with rabbit-anti-HA1 antibody (1:2000, ImmuneTechnology, USA) for detection of HA and HA1. As for lane 4, themembrane was incubated with rabbit-anti-NA antibody (1:2000, ImmuneTechnology, USA) for detection of NA. The HRP labeled goat anti-rabbitIgG antibody (1:2000, Invitrogen, USA) was used as the second antibodyto detect the bindings in lanes 2-4. The results showed that viralproteins HA and HA1 were recognized by both the P9 (SEQ ID NO: 10) (lane2) and rabbit-anti-HA1 antibodies (lane 3), but the P9 (SEQ ID NO: 10)could not bind to NA (lane 2) which was recognized by rabbit-anti-NAantibodies (lane 4). The results of both Western blot assay (FIG. 6B)and ELISA (FIG. 6C) showed that the P9 (SEQ ID NO: 10) bound to viralsurface glycoprotein HA but not NA. Thus, it was concluded that the P9(SEQ ID NO: 10) can bind to viral surface protein HA to inhibit thevirus infection.

Experimental Example 4: The P9 (SEQ ID NO: 10) Blocked Viral Disassemblyfor RNA Release from Late Endosomes

It was further investigated which step or steps of the virus infection,including virus-receptor binding, endocytosis and virus-endosomemembrane fusion to release the viral RNA, had been involved by theP9-mediated inhibition. The Dio dye labeled H1N1 virus was pretreatedwith 25 μg/ml of the P9 (SEQ ID NO: 10) (indicated by P9 in FIG. 7A) orPB (indicated by VC in FIG. 7A) for 1 hour. The virus (20 MOI) wasinoculated to MDCK cells and incubated at 4° C. for 3 hours or 5 MOI ofthe virus was inoculated to the MDCK cells and incubated at 37° C. for 1hour or 2 hours. The infected cells were fixed and cell membrane wasstained by Alex-594 dye. The results showed that when thefluorescence-labeled virus was pretreated with the P9 (SEQ ID NO: 10)and then incubated with the cells at 4° C., the P9 (SEQ ID NO: 10) couldnot block the virus binding to the cell membrane (FIG. 7A). FIG. 7A alsoshowed that the pretreatment of the labeled virus with the P9 (SEQ IDNO: 10) did not affect the virus entry into cells by endocytosis afterincubation at 37° C. for 1 hour, and the pretreatment of the labeledvirus with the P9 (SEQ ID NO: 10) did not affect the maturation of theendosomes after incubation at 37° C. for 2 hours, in which the lateendosome had moved to near nucleus. In FIG. 7B, the Dio dye labeled H1N1virus was pretreated with 50 μg/ml of the P9 (SEQ ID NO: 10) for 1 hour,and then MDCK cells were infected with the virus and incubated at 37° C.for 2 hours. Then, the cells were fixed by 10% (v/v) formalin in PBSbuffer and stained with rabbit-anti-smBD4 antibody and Alexa594 labeledgoat-anti-rabbit antibody. FIG. 7B showed that P9 (SEQ ID NO: 10) couldbe delivered to the late endosomes by binding to the virus and moved tothe perinuclear region together with the virus at 2 hours after viralinfection. Furthermore, as shown in FIG. 7C, the viral RNA levels in thecells infected by the P9-pretreated virus decreased after the infection,reaching over 2 folds lower than that in the untreated virus control at3.5 hours post-infection. In contrast, the viral RNA in untreated virusculture maintained at similar levels during the first 2.5 hourspost-infection and started to increase at 3.5 hours post-infection,suggesting that the viral RNA had been transported through nuclear poreto nucleus and initiated a new viral replication. These resultsindicated that the P9 (SEQ ID NO: 10) could not block the virus-receptorbinding and virus entry into the cells by endocytosis. Instead, theresults suggested that P9 (SEQ ID NO: 10) inhibited viral RNA releasefrom late endosomes.

Experimental Example 5: The P9 (SEQ ID NO: 10) Might Suppress the pHDecrease (Acidification) in Endosomes to Block the Virus-EndosomeMembrane Fusion and Viral Disassembly for RNA Release

In consideration of the rich basic amino acids in the P9 (SEQ ID NO:10), it has been further investigated whether the P9 (SEQ ID NO: 10)could inhibit virus-endosome membrane fusion by suppressing the decreaseof pH in late endosomes. The H1N1 virus was pretreated with PB(indicated by VC in FIG. 8A) or the P9 (SEQ ID NO: 10) (50 μg/ml)(indicated by P9 in FIG. 8A), and 5 MOI of the virus was inoculated toMDCK cells and incubated at 37° C. Viral RNA copies in cell samplescollected at indicated time-points after inoculation were detected byreal-time RT-PCR and compared with those from the infected cells in thepresence of 10 mM and 0.1 mM of a well-defined endosome inhibitor NH₄Cl.As shown in FIG. 8A, the P9 (SEQ ID NO: 10) showed a similar inhibitoryeffect to virus infection as compared to that treated with optimalinhibitory concentration (10 mM) of a well-defined late endosomalinhibitor ammonium chloride (NH₄Cl) (see “Lakadamyali, M., M. J. Rust,H. P. Babcock, and X. Zhuang. 2003. Visualizing infection of individualinfluenza viruses. Proc Natl Acad Sci USA 100:9280-9285” and “Matlin, K.S., Reggio, H., Helenius, A. & Simons, K. Infectious entry pathway ofinfluenza virus in a canine kidney cell line. J Cell Biol 91, 601-613(1981)”). The inhibitory effect of the P9 (SEQ ID NO: 10) to the viralRNA reached the highest level at 3.5 hours post-infection, in which theviral RNA copies obtained from the P9-pretreated virus was even a littlelower than that treated with 10 mM NH₄Cl. The inhibitory effect of theP9 (SEQ ID NO: 10) to the viral RNA reached similar level to thattreated with 10 mM NH₄Cl at 6.5 hours post-infection.

To further confirm the inhibitory activity of the P9 (SEQ ID NO: 10) inendosomal acidification, the inventors have detected the pH decrease inendosomes using pH-sensitive dye (FIG. 8B). When the cells were infectedwith untreated virus (indicated by VC), red spots (in color image,corresponding to the gray spots in black and white image, as seen in theupper left picture) in the infected cells indicated the acidificationprocess in late endosomes, since the pH-sensitive dye is a pH indicatorwhich would show red fluorescence when pH value dropped down to 5 from7. White arrows in the pictures point to the viral locations (greenspots in color image, corresponding to the gray spots in black and whiteimage, as seen in the lower left picture of FIG. 8B) and theircorresponding endosomal acidification (red spots in color image,corresponding to the gray spots in black and white image, as seen in theupper left picture of FIG. 8B). In contrast, when the cells wereinfected with P9-pretreated virus (indicated by P9 (SEQ ID NO: 10)), nored fluorescence in color image was observed (see the upper rightpicture of FIG. 8B), indicating that no acidification process occurredin the endosomes. The results have clearly demonstrated that the P9 (SEQID NO: 10) could prevent acidification in endosomes when it wasdelivered into the endosomes together with the virus.

Experimental Example 6: Basic Amino Acids in the P9 (SEQ ID NO: 10) Playa Key Role in the Antiviral Activity

To define whether the rich basic amino acids in the P9 (SEQ ID NO: 10)played the key role in suppressing the pH decrease in late endosomes,P9-analogous peptides were designed and synthesized by substituting 1 to3 basic amino acids with neutral or acidic amino acids at C-terminus oradding 3 acidic amino acids or 3 basic amino acids at N-terminus of theP9 (SEQ ID NO: 10) (Table 3). The binding affinities of these analogouspeptides to viral protein HA were detected by ELISA (FIG. 9A). Theresults showed that the reduction of 1 and 2 basic amino acids (P9-S1(SEQ ID NO:13) and P9-S2 (SEQ ID NO:14)) and addition of 3 acidic aminoacids (P9-aci-1) (SEQ ID NO:16) or 3 basic amino acids (P9-KHR) (SEQ IDNO:17) did not affect the binding affinity of these P9-analogouspeptides. The reduction of 3 basic amino acids (P9-S3 (SEQ ID NO:15))resulted in significant decrease of binding affinity to viral proteinHA. In addition, inhibitory activities of the peptides againsthemagglutination of viral HA protein were measured by HAI assay (FIG.9B). The indicated peptides (50 μg/ml) were 2-fold serially diluted. Thediluted peptides and PBS (Invitrogen, USA) (negative control) werepre-incubated with H1N1 virus for 1 hour at room temperature and then 50μl of 5% (v/v) turkey red blood cells (TRBC) were added to each well.The results were observed when the PBS control appeared typicalhemagglutination and the highest dilutions (HAI activity) of thepeptides that still inhibited the hemagglutination were recorded. Theresults indicated that both P9-S1 (SEQ ID NO:13) and P9-aci-1 (SEQ IDNO:16) exhibited HAI activity similar to that of the P9 (SEQ ID NO: 10)(FIG. 9B) (the dilution folds are indicated on the vertical axis).Furthermore, antiviral effects of the P9-analogous peptides in MDCKcells were detected using real-time RT-PCR. The virus (50 PFU/well) waspre-incubated with the indicated peptides (50 μg/ml) or PB (indicated byVC in FIG. 9C) for 1 hour at RT and then inoculated to MDCK cells. Theinfected cells were harvested at indicated time-points after inoculationand the viral RNA copies were detected by real-time RT-PCR. Data werepresented as mean+SD of three independent experiments. The resultsshowed that reduction of 1 or 2 basic amino acids (P9-S1 (SEQ ID NO:13)and P9-S2 (SEQ ID NO:14)) and addition of 3 acidic amino acids(P9-aci-1) (SEQ ID NO:16) resulted in decrease of antiviral effects ofthese P9-analogous peptides (FIG. 9C). However, addition of 3 basicamino acids at N-terminus of the P9 (P9-KHR) (SEQ ID NO:17) did notresult in decrease of its antiviral effect. These results elucidatedthat enriched basic amino acids in the P9 (SEQ ID NO: 10) indeed playeda critical role in the inhibition of viral infection.

TABLE 3 Five P9-analogous peptides were designed and synthesized Peptidename Sequence SEQ ID No. smBD4 iinnpitcmtngaicwgpcptafrqigncghfkvrcckir1 P9-S1 ngaicwgpcptafrqigncghfkvrccnir 13 P9-S2ngaicwgpcptafrqigncghfkvrccnid 14 P9-S3 ngaicwgpcptafrqigncghfkvtccnid15 P9-aci-1 dedngaicwgpcptafrqigncghfkvrcckir 16 P9-KHRkhrngaicwgpcptafrqigncghfkvrcckir 17 Notes: The changed amino acids werebolded. P9-S1 (SEQ ID NO: 13): one basic amino acid K was replaced by N.P9-S2 (SEQ ID NO: 14): two basic amino acids K and R were replaced by Nand D. P9-S3 (SEQ ID NO: 15): three basic amino acids R, K and R werereplaced by T, N and D. P9-aci-1 (SEQ ID NO: 16): three acidic aminoacids D, E and D were added at N-terminus of the P9 (SEQ ID NO: 10).P9-KHR (SEQ ID NO: 17): three basic amino acids K, H and R were added atN-terminus of the P9 (SEQ ID NO: 10).

Experimental Example 7: The P9-Mediated Antiviral Effect Also Related toits High Binding Affinity to Viral Glycoprotein HA

Another peptide P8 (SEQ ID NO: 9) is a shorter form of the P9 (SEQ IDNO: 10) that contains 20 amino acids of the C-terminal region of P9 (SEQID NO: 10) but all 6 basic amino acids are the same as the P9 (Table 1).However, it showed much lower antiviral effect compared to the P9 (SEQID NO: 10) (FIGS. 2A and 9C). The binding affinity of the P8 (SEQ ID NO:9) to viral protein HA was detected using ELISA (FIG. 9A), and HAI wasdetected (FIG. 9B). The results showed that the P8 (SEQ ID NO: 9)exhibited significantly lower binding affinity to HA than that of the P9(SEQ ID NO: 10). Thus, the antiviral efficacy of such kind of antiviralpeptides may be determined by not only the number of basic amino acidsin the peptides but also their binding affinity to the viruses.

Experimental Example 8: The P9 (SEQ ID NO: 10) Showed Broad SpectrumAntiviral Efficacies Against Respiratory Viruses

The inventors further detected whether the P9 (SEQ ID NO: 10) could alsoinhibit infections of the other enveloped respiratory viruses whichenter target cells through the endosomal pathway, e.g. the othersubtypes of influenza A virus, SARS-CoV and MERS-CoV, in cell cultures.Influenza virus subtypes H3N2, H5N1, H7N7 and H7N9 were pretreated withthe serially diluted P9 (SEQ ID NO: 10) for 1 hour at RT and theninoculated to MDCK cells. The inhibitory effects of the P9 (SEQ ID NO:10) against infections of these viruses were determined by plaque assay.As shown in FIG. 10(a), the P9 (SEQ ID NO: 10) exhibited strongantiviral efficacies against infections of influenza A virus subtypesH3N2, H5N1, H7N7 and H7N9. The IC₅₀s of the P9 (SEQ ID NO: 10) againstinfections of these subtypes of influenza A virus ranged from about 1.5to 4.8 μg/ml, which were a little higher than that against H1N1influenza virus (FIG. 2B).

In addition, inhibitory effects of the P9 (SEQ ID NO: 10) againstinfections of SARS-CoV and MERS-CoV were detected by plaque assay inFRhK4 and Vero E6 cells. Notably, antiviral efficacies against SARS-CoVand MERS-CoV of the P9 (SEQ ID NO: 10) were higher when itsconcentrations were over 25 μg/ml but lower when its concentrations werelower than 25 μg/ml, as compared to its antiviral efficacies againstinfluenza A virus (FIG. 10(b)). The IC₅₀s of P9 (SEQ ID NO: 10) againstSARS-CoV and MERS-CoV were about 10 μg/ml, which were about 2-7 foldshigher than that against influenza A virus. These results indicated thatP9 (SEQ ID NO: 10) have broad-spectrum antiviral activities againstmultiple respiratory viruses which infect target cells through theendosomal pathway.

CONCLUSION

Although many defensins from murine or humans have been found to haveantiviral activity in vitro and in vivo (see “Sun, L., et al. Humanbeta-defensins suppress human immunodeficiency virus infection:potential role in mucosal protection. J Virol 79, 14318-14329 (2005)”,“Quinones-Mateu, M. E., et al. Human epithelial beta-defensins 2 and 3inhibit HIV-1 replication. Aids 17, F39-48 (2003)” and “Jiang, Y., etal. Expression of mouse beta-defensin-3 in MDCK cells and itsanti-influenza-virus activity. Arch Virol 154, 639-647 (2009)”), thepresent invention first reported that the constructed recombinant mouseβ-defensin-4 according to the present disclosure (FIG. 1) has strongantiviral effects against infections of broad respiratory viruses.However, the development of defensins as systemic therapeutics has beenhindered by several factors, such as suboptimal efficacy, side effectsand the lack of cost-effective means of commercial-scale production (see“Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature415, 389-395 (2002)”).

In the present invention, in order to solve the above-mentionedproblems, 11 short peptides derived from mBD4 were designed andsynthesized. After screening antiviral efficacies of these shortpeptides, it has been found that one peptide P9 (SEQ ID NO: 10), whichis a short form of mBD4 containing 30 amino acids at C-terminus of mBD4(Table 1), exhibited the highest antiviral activity against influenza Avirus H1N1 in vitro, reaching an IC₅₀ of about 1.2 μg/ml which was lowerthan that of smBD4 (SEQ ID NO: 1) and rmBD4 (FIG. 2B). Furthermore, itis surprisingly found that the P9 (SEQ ID NO: 10) showed much morepotent prophylactic and therapeutic effects against lethal infection ofthe H1N1 virus than rmBD4 in a mouse model (in vivo) (FIGS. 3 and 4).Compared to smBD4 (SEQ ID NO: 1) and rmBD4, the P9 (SEQ ID NO: 10) alsoshowed the lowest cytotoxicity (FIG. 2C and Table 2) and has moreexcellent selectivity index which was about 25 and 2 folds higher thanthat of smBD4 (SEQ ID NO: 1) and rmBD4, respectively. Although it hasbeen reported that some short peptides derived from human β-defensin-3exhibited more potent antibacterial effects and less toxicity to hostcells than β-defensin-3 itself (see “Bai, Y., et al. Structure-dependentcharge density as a determinant of antimicrobial activity of peptideanalogues of defensin. Biochemistry 48, 7229-7239 (2009)”), this is thefirst disclosure that short peptides derived from β-defensin can providemuch more excellent selectivity index and potent antiviral effects invivo. The smBD4 (SEQ ID NO: 1) cannot be a candidate of antiviral drugbecause of its high cytotoxicity. The production (including expression,enzyme-treatment and purification) of recombinant β-defensins istime-consuming and expensive, which also limited the development ofβ-defensins as antiviral agents. In contrast, the peptides of thepresent invention not only exhibited the highest antiviral activity andlowest cytotoxicity, but also can be directly synthesized using achemical method and is highly soluble in water. Thus, the peptides ofthe present invention are ideal candidates for development of novelantiviral drugs.

To understand antiviral mechanism of the peptides of the presentinvention, the inventors first demonstrated that the P9 (SEQ ID NO: 10)inhibited the virus infection but not virus replication or release(FIGS. 5A, 5D, and 5G). Since it has been reported that the antiviralactivity of β-defensins might be mediated through either indirectinteraction with target cells infected by virus or direct interactionwith viral glycoproteins and/or envelopes (see “Klotman, M. E. & Chang,T. L. Defensins in innate antiviral immunity. Nat Rev Immunol 6, 447-456(2006)” and “Ding, J., Chou, Y. Y. & Chang, T. L. Defensins in viralinfections. J Innate Immun 1, 413-420 (2009)”), the inventors thendetermined whether the P9 (SEQ ID NO: 10) bound to the virus or thetarget cells. The results showed that the P9 (SEQ ID NO: 10) bound tovirus (FIGS. 5C, 5F, 5I and 6A) but not the cell membrane (FIGS. 5B, 5E,5H and 6A) to inhibit the virus infection. The present invention furtherdefined that the P9 (SEQ ID NO: 10) bound to glycoprotein HA but not theother viral protein NA on the viral surface (FIGS. 6B and 6C). It hasbeen well-known that the infection of influenza virus goes throughmultiple steps, i.e., virus-receptor binding, endocytosis, movement fromearly endosomes to late endosomes where endosomal acidification resultsin virus-endosome membrane fusion and subsequent viral disassembly(uncoating) for the release of viral RNA to trigger viral replication(see “Lakadamyali, M., M. J. Rust, H. P. Babcock, and X. Zhuang. 2003.Visualizing infection of individual influenza viruses. Proc Natl AcadSci USA 100:9280-9285” and “Leikina, E., H. Delanoe-Ayari, K. Melikov,M. S. Cho, A. Chen, A. J. Waring, W. Wang, Y. Xie, J. A. Loo, R. I.Lehrer, and L. V. Chernomordik. 2005. Carbohydrate-binding moleculesinhibit viral fusion and entry by crosslinking membrane glycoproteins.Nat Immunol 6:995-1001”). Thus, the present invention furtherinvestigated which step was involved by the P9-mediated inhibitoryeffect. The results indicated that the P9 (SEQ ID NO: 10) could notinhibit the virus-receptor binding and endocytosis but inhibited viraldisassembly and viral RNA release from the late endosomes (FIG. 7).

The next question which should be addressed is that why and how thepeptides of the present invention could inhibit viral disassembly andviral RNA release from the late endosome. Low pH (5.0) in the lateendosome is critical for influenza virus-endosome membrane fusion.Previous study has demonstrated that a late endosomal inhibitor NH₄Clcan inhibit virus-endosome membrane fusion by inhibiting the decrease ofpH in late endosomes (see “Matlin, K. S., Reggio, H., Helenius, A. &Simons, K. Infectious entry pathway of influenza virus in a caninekidney cell line. J Cell Biol 91, 601-613 (1981)” and “Lakadamyali, M.,Rust, M. J., Babcock, H. P. & Zhuang, X. Visualizing infection ofindividual influenza viruses. Proc Natl Acad Sci USA 100, 9280-9285(2003)”). Considering that the P9 (SEQ ID NO: 10) is rich of basic aminoacids, the inventors have tested whether the P9 (SEQ ID NO: 10) couldalso exert the antiviral function by inhibiting the pH decrease in lateendosomes and, in turn, blocking the virus-endosome membrane fusion andsubsequent viral RNA release into nucleus to trigger the virusreplication. The results demonstrated that the P9 (SEQ ID NO: 10)exhibited similar inhibitory effect as NH₄Cl to inhibit viral RNArelease and the inhibitory effect thereof reached the highest level at3.5 hours post-infection (FIG. 8A). It was also shown that the P9 (SEQID NO: 10) could indeed inhibit the decrease of pH in late endosomes(FIG. 8B). Furthermore, the basic amino acids in P9 (SEQ ID NO: 10) wereindispensable for the suppression of progressive acidification in lateendosomes. When 3 additional acidic amino acids were added to the P9(P9-aci-1) (SEQ ID NO:16) or 1 to 2 basic amino acids were substituted(P9-S1 (SEQ ID NO:13) and P9-S2 (SEQ ID NO:14)), antiviral effects ofthese P9-analogous peptides decreased (FIG. 9C), although their bindingaffinity was similar or even higher than that of the P9 (SEQ ID NO: 10)(FIG. 9A). Thus, the results have demonstrated that the basic aminoacids contained in such kind of antiviral peptides may play a key rolein suppressing pH decrease in the late endosomes.

Notably, another important factor which may affect antiviral activity ofsuch peptides is the binding affinity of the peptides to the virus. Thiswas elucidated by the fact that the P8 (SEQ ID NO: 9) showed lowerbinding affinity (FIGS. 9A and 9B) and antiviral effect (FIGS. 2A and9C) than those of the P9 (SEQ ID NO: 10), although it contains the samenumber of basic amino acids as the P9 (SEQ ID NO: 10) (Table 1). Acommon feature of most AMPs is their net-positive charge which triggersthe initial interaction between AMPs and microbe membrane protein orphospholipids with negative charge (see “Jung, S., et al. Humanbeta-defensin 2 and beta-defensin 3 chimeric peptides reveal thestructural basis of the pathogen specificity of their parent molecules.Antimicrob Agents Chemother 55, 954-960 (2011)” and “Jenssen, H.,Hamill, P. & Hancock, R. E. Peptide antimicrobial agents. Clin MicrobiolRev 19, 491-511 (2006)”). In addition, the antimicrobial activity ofcationic peptides is also related to their hydrophobicity (see“Kustanovich, I., Shalev, D. E., Mikhlin, M., Gaidukov, L. & Mor, A.Structural requirements for potent versus selective cytotoxicity forantimicrobial dermaseptin S4 derivatives. J Biol Chem 277, 16941-16951(2002)” and “Zelezetsky, I., Pag, U., Sahl, H. G. & Tossi, A. Tuning thebiological properties of amphipathic alpha-helical antimicrobialpeptides: rational use of minimal amino acid substitutions. Peptides 26,2368-2376 (2005)”) and their secondary structure (see “Jenssen, H.,Hamill, P. & Hancock, R. E. Peptide antimicrobial agents. Clin MicrobiolRev 19, 491-511 (2006)”). The lower binding affinity of the P8 (SEQ IDNO: 9) may be related to the deletion of 10 amino acids on N-terminus,which could probably affect the hydrophobicity or secondary structure ofthe P8 (SEQ ID NO: 9), resulting in decrease of binding affinity of theP8 (SEQ ID NO: 9).

For countless pathogen viruses, including influenza viruses andcoronaviruses, viral disassembly and viral RNA releasing into host cellsto initiate a productive viral replication requires fusion of viral andendosome membranes (see “Smith, A. E. & Helenius, A. How viruses enteranimal cells. Science 304, 237-242 (2004)” and “Du, L., et al. The spikeprotein of SARS-CoV-a target for vaccine and therapeutic development.Nat Rev Microbiol 7, 226-236 (2009)”). Membrane fusion is mediated byviral envelope glycoproteins (for example, HA of influenza virus or Sprotein of SARS-CoV) and needs an acidic environment in late endosomes(see “Du, L., et al. The spike protein of SARS-CoV—a target for vaccineand therapeutic development. Nat Rev Microbiol 7, 226-236 (2009)” and“Das, K., Aramini, J. M., Ma, L. C., Krug, R. M. & Arnold, E. Structuresof influenza A proteins and insights into antiviral drug targets. NatStruct Mol Biol 17, 530-538 (2010)”). In the present invention, theinventors have illustrated that the mechanism of antiviral effectmediated by the peptides of the present invention is related to two keyfactors. The first is that the peptides can bind to the surfaceglycoprotein of the virus, and the second is that the peptides mayinhibit pH decrease (acidification) in late endosomes to block thevirus-endosome membrane fusion and subsequent viral RNA release. Basedon this mechanism, the peptides of the present invention should be alsoable to inhibit infections of other countless pathogen viruses. Theresults showed that the P9 (SEQ ID NO: 10) could indeed inhibitinfections of the other subtypes of influenza A virus including H3N2,H5N1, H7N7 and H7N9 (FIG. 10(a)) and two coronaviruses SARS-CoV andMERS-CoV (FIG. 10(b)). It is reasonable to predict that the peptides ofthe present invention are capable of inhibiting infections of othercountless pathogen viruses if they can efficiently bind to the surfaceglycoprotein of these viruses. Thus, the peptides of the presentinvention are ideal candidates to be developed as broad-spectrumantiviral drugs against infections of countless pathogen viruses,particularly those respiratory viruses.

In summary, it has been demonstrated that a short peptide derived frommBD4 may provide broad-spectrum antiviral activities against infectionsof different subtypes of influenza A virus including H1N1, H3N2, H5N1,H7N7 and H7N9, as well as two coronaviruses SARS-CoV and MERS-CoV. Themechanism of the broad-spectrum antiviral effects of the peptides of thepresent invention is elucidated to be due to: (1) the peptides canefficiently bind to viral glycolproteins on the surface of the viralparticles; and (2) the peptides contain rich basic amino acids which caninhibit pH decrease (acidification) in late endosomes to block thevirus-endosome membrane fusion and subsequent viral disassembly andviral RNA release. Influenza A virus and coronavirus have caused severalfatal pandemics and outbreaks in recent century. Although severalanti-influenza drugs have been developed, drug-resistant virus strainsemerged quickly after these antiviral drugs applied for the clinicaltreatment. Particularly, thus far, no any effective antiviral drug maybe used for prophylaxis and therapy against infections of SARS-CoV andMERS-CoV yet. In this regard, antiviral peptides like the P9 (SEQ ID NO:10) may be new promising prophylactic and therapeutic agents withbroad-spectrum antiviral activities and low possibility to result indrug resistance. Moreover, based on the mechanism illustrated in thisdisclosure, more new antiviral peptides with broad-spectrum antiviralactivities may be designed and developed to new commercial antiviralagents for prophylaxis and therapy of countless pathogen viruses.

It would be appreciated by those skilled in the art that various changesand modifications may be made to the described exemplary embodimentswithout departing from the spirit and essential of the invention. Thosechanges and modifications fall into the scope of the appended claims.

What is claimed is:
 1. A method of reducing the likelihood of developingrespiratory virus infections, including a step of administering to asubject in need thereof an effective amount of a peptide synthesizedthrough a chemical route or by a genetic engineering process,characterized in that the peptide has a functional domain capable ofbinding to a surface glycoprotein of a respiratory virus and has anactivity of inhibiting infection of the respiratory virus, wherein thepeptide has 5 or more basic amino acids, among which 2 or more basicamino acids are in N-terminal region or C-terminal region of thepeptide; and wherein the N-terminal region comprises a sequence of nomore than 10 amino acids counting from the N-terminal amino acid of thepeptide, and the C-terminal region comprises a sequence of no more than10 amino acids counting from the C-terminal amino acid of the peptide;and wherein the peptide consists of an amino acid sequence that is atleast 90% identical to SEQ ID NO:
 10. 2. The method according to claim1, wherein the peptide has a function of preventing acidification in alate endosome of a cell.
 3. The method according to claim 1, wherein thepeptide has 3 or more basic amino acids in the N-terminal region orC-terminal region thereof.
 4. The method according to claim 3, whereinthe peptide has 4 or more cysteines.
 5. The method according to claim 1,wherein the amino acid sequence of the peptide consists of SEQ ID NO:10, SEQ ID NO: 13, or SEQ ID NO:
 17. 6. The method according to claim 1,wherein the C-terminal region has two cysteines and the basic aminoacids.
 7. The method according to claim 6, wherein the C-terminal regionhas 10 amino acids with the following amino acid composition: basicamino acid-neutral amino acid-basic amino acid-neutral amino acid-basicamino acid-cysteine-cysteine-basic amino acid-neutral amino acid-basicamino acid-free carboxyl.
 8. The method according to claim 1, whereinthe peptide is originated from mouse β-defensin-4.
 9. The methodaccording to claim 1, wherein the respiratory virus is selected frominfluenza viruses and coronaviruses; wherein the influenza virus isselected from the group consisting of subtypes H1, H3, H5, and H7, andthe coronavirus is selected from the group consisting of SARS-CoV andMERS-CoV.